The present invention relates to novel variants of growth hormone releasing hormone (GHRH) that have enhanced resistance to enzymatic degradation and polynucleotides encoding said GHRH variants. The present invention relates to methods and therapeutic compositions for the treatment of growth hormone related deficiencies comprising administrating to humans, companion animals, livestock or poultry, plasmid compositions comprising polynudeotides encoding GHRH or variants thereof, alone or in combination with polynucleotides encoding growth hormone or modified growth hormone. The present invention further relates to methods and compositions that promote the release and expression of growth hormone in order to enhance the growth and performance of companion animals, livestock or poultry comprising the administration plasmid compositions encoding GHRH variants, GHRH or modified GHRH, and/or GH or modified GH.
Growth hormone-releasing hormone (xe2x80x9cGHRHxe2x80x9d) is a peptide hormone secreted from the hypothalamus. Following secretion, GHRH enters the portal circulation connecting the hypothalamus to pituitary gland. GHRH then interacts with its receptors on the pituitary gland and induces the release of growth hormone (xe2x80x9cGHxe2x80x9d). GH secreted from the pituitary gland enters the general circulation and from there it reaches various organs and tissues of the body where it interacts with specific receptors and induces a wide range of developmental effects.
GHRH peptides have been isolated and characterized from several species including humans, porcine, ovine and bovine. In each of these species, GHRH is a small polypeptide consisting of 44 amino acids (GHRH(1-44)-NH2). However, it has been also shown that smaller fragments, most notably those consisting of the first (amino terminal) 29 amino acids (referred to as GHRH1-29 fragment) retain the same intrinsic biological activity as the full length parent molecule.
GHRH is synthesized as a precursor polypeptide consisting of 107 or 108 amino acids depending on the species. Following synthesis, the precursor GHRH polypeptide undergoes sequential processing. First, the 31 amino acid signal peptide (Metxe2x88x9230 to Arg0) of the GHRH precursor polypeptide is cleaved (Smith et al., 1992, Biotechnology 10:315-319). Subsequently, the GHRH precursor polypeptide is cleaved at position 46-47 and at position 45-46 by a trypsin-like endopeptidase and a carboxypeptidase, respectively, resulting in generation of GHRH(1-45)-OH and a 30 amino acid peptide (amino acids 77-107) designated GCTP (Brar, A. K. et al., 1991, Endocrinology 129: 3274-3280). The GHRH(1-45)-OH polypeptide is further processed by peptidyl glycine xcex1-amidating monooxygenase (xe2x80x9cPAMxe2x80x9d), which transfers an amide group from Gly45 to Leu44 and results in the formation of GHRH(1-44)-NH2, the full length form of GHRH (Brar, A.K. et al., 1991, Endocrinology 129: 3274-3280). The GCTP is also undergoes processing by PAM, which results in the transfer of an amide group from Gly77 to Gln76. Although the role of GHRH(1-44)-NH2 in inducing the release of GH is well established, the role of the GCTP peptide is less clear. One report has implicated the GCTP peptide in the control of feeding behavior (Arase, K. et al., 1987, Endocrinology 121:1960-1965).
GH has been identified and its gene cloned from many species including human, porcine, bovine, and equine. Unlike GHRH, there exists natural variants of GH within a given species. For example, bovine GH is released from the pituitary gland in one of four variants which differ from one another by one or more amino acids and some studies suggest that these variants differ in their potency (e.g., in terms of their ability to increase milk yield). Several studies have also identified amino acid substitutions that lead to an increase in the affinity of GH to its receptors and/or enhanced stability to enzymatic degradation. Studies have also shown that immunization against specific peptides from GH (e.g., a peptide consisting of amino acids 35 to 53 of GH) leads to production of antibodies that bind growth hormone and increase the efficacy of GH treatment, presumably because the antibodies delay the clearance of GH from circulation, thus, increasing half-life of GH, and/or protect GH from proteolytic degradation (Bomford, F. and Aston, P., 1990, Endocnnology 125:31-38).
Significant research efforts have focused on the structural attributes of GH and GHRH, as well as their biological and developmental activities. A number of groups have attempted to exploit GH and GHRH in a manner that could provide important therapeutic and economic benefits as a result of their use in humans and animals. For example, the traditional treatment of GH-deficient children has been the administration of growth hormone isolated from human pituitary glands, however these preparations are no longer available in the United States due to virus-contaminated samples (Vance, 1990, Clin. Chem 36/3: 415-420). Recombinantly expressed and purified GH have been shown to have some benefits in treating GH-deficient children, however the combination of recombinantly expressed GH and GHRH in the treatment of GH-deficient children has provided conflicting results. (Vance. supra). Further, purified GH and GHRH must be administered at very high quantities to be effective as the exposure of these polypeptides to serum results in their rapid degradation to a polypeptide which exhibits considerably different biological and pharmacokinetic properties. (Fronman et al., 1989, J. Clin. Invest. 83:1533-1540).
Other studies have shown that GH or GHRH administered as purified polypeptides have significant impact on animal growth (muscle and bone growth), average daily gain, milk production, feed efficiency (the ratio of feed consumed to body weight gain), adipose tissue accretion and others. For example, it has been shown that daily administration of maximally effective doses of GH administered to growing pigs for 33-77 days can increase average daily gain xe2x88x9210-20%, improves feed efficiency 13-33%, decrease adipose tissue accretion by as much as 70%, and stimulates protein deposition (muscle growth) by as much as 62%. (Etherton et al.,1998, Physiological Reviews 78:745-761). Furthermore, when GH was administered to dairy cows, milk yields were increased by 10-15% (xe2x96xa14-6kg/day) (Etherton et al.,1998, Physiological Reviews 78:745-761).
A major impediment to fulfilling the therapeutic and economic potential of GHRH peptides is their susceptibility to cleavage (and subsequent conversion to inactive forms) by specific tissue and plasma proteolytic enzymes; most notably dipeptidylpeplidase IV (xe2x80x9cDPPIVxe2x80x9d). A number of researchers have focused on manipulating GHRH in order to develop compounds with significant therapeutic potential. Consequently, a wide variety of synthetic GHRH peptide analogues have been produced. They consist of GHRH polypeptides in which one or more amino acids have been chemically modified or replaced with other L- or D-amino acids. These modifications or substitutions are designed to yield analogues with biological properties superior to those of the parent molecule in terms of potency, stability and resistance to chemical and enzymatic degradation. However, these chemically modified polypeptides are not easily or efficiently produced in a suitable form to be administered to humans or animals.
In spite of the significant therapeutic and economic benefits of GH or GHRH alluded to above, exogenous supplementation of animals with GH or GHRH proteins have not been widely adopted as a component of routine management practices to enhance the quality of meat from animals and/or enhance the productivity of livestock. This is because in order to get these benefits, animals have to be repeatedly administered GH or GHRH polypeptides (often daily, but typically in a slow release formulation given every 7-10 days). (Etherton, T. D., 1997, Nature Biotechnology 15:1248) This situation is labor intensive, time consuming, expensive, and does not fit current management practices where animals are reared in large numbers and are handled very infrequently, it is apparent therefore that in order to realize the therapeutic and economic benefits of GH and/or GHRH administration, much improved formulations for delivery of these hormones must be developed to overcome the current limitations of their use; namely the need for repeated administration.
The present invention relates to novel variants of GHRH that have enhanced resistance to enzymatic degradation and polynucleotides encoding said variants. The present invention also relates to pharmaceutical formulations comprising polynucleotide sequences encoding GHRH variants alone or in combination with polynucleotide sequences encoding GHRH, modified GHRH, GH and/or modified GH. The present invention also relates to pharmaceutical formulations comprising GHRH variant peptides alone or in combination with GHRH polypeptides, modified GHRH polypeptides, GH polypeptides and/or modified GH polypeptides. The present invention further relates to pharmaceutical formulations comprising canine or feline GHRH peptides alone or in combination with GHRH variant polypeptides, modified GHRH polypeptides, GH polypeptides and/or modified GH polypeptides.
The present invention relates to therapeutic methods and compositions for the treatment of growth hormone related deficiencies comprising growth hormone (xe2x80x9cGHxe2x80x9d) and/or growth hormone-releasing hormone (xe2x80x9cGHRHxe2x80x9d) in human, companion animals, livestock and poultry. The invention also relates to methods for the improvement in the health of humans, companion animals, livestock and poultry. The invention also relates to methods for the treatment of obesity and frailty of companion animals. The invention further relates to methods for the enhancement of the growth and performance of companion livestock and poultry. The methods of the present invention comprise pharmaceutical compositions which enhance the expression of growth hormone or promote the release of growth hormone or both when administered to humans, companion animals, livestock or poultry. According to the present invention, the term xe2x80x9cGHRHxe2x80x9d relates to the full length wildtype form of GHRH which is 44 amino acids (aa) or a precursor form of GHRH. In accordance with the present invention, the term xe2x80x9cmodified GHRHxe2x80x9d refers to any amino terminal polypeptide fragment of GHRH from 29 amino acids to 107 or 108 amino acids in length and any mutant of GHRH, including additions, deletions or substitutions at the nudeotide or amino acid level, which retains at least the level of activity of wildtype GHRH, that is, the ability to induce GH gene transcription at levels comparable to wildtype GHRH.
In accordance with the present invention, the term xe2x80x9cGHRH variantxe2x80x9d relates to a GHRH polypeptide to which one or more amino acids have been attached to the carboxy or amino terminus of the polypeptide, or a wildtype GHRH polypeptide that contains a substitution of one or more amino acids, so that the GHRH variant retains at least equal or enhanced wildtype GHRH activity and has enhanced resistance to enzymatic degradation relative to the wildtype GHRH. In accordance with the present invention, wildtype GHRH activity is measured by its ability to induce GH gene transcription. In accordance with the present invention, resistance to enzymatic degradation is determined by the ability of the polypeptide to resist degradation caused by dipeptidylpeptidase type IV.
According to the present invention, the term xe2x80x9cGHxe2x80x9d refers to the full length wildtype form of GH, which is 191 amino acids, and xe2x80x9cmodified GHxe2x80x9d refers to any fragment of GH and any mutant including additions, deletions or substitutions at the nucleotide or amino acid level, which retains at least the level of wildtype activity of GH, that is, the ability to induce insulin growth factor (IGF) gene transcription at levels comparable to wildtype GH, or mimic the anti-adipogenic and lipolytic effects of GH.